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The program for in vitro fertilization at Norfolk*
Vol. 38, No.1, July 1982
FERTILITY AND STERILITY Copyright 0 1982 The American Fertility Society
Printed in U.SA.
The program for in vitro fertilization at Norfolk*
Howard W. Jones, Jr., M.D.t:J: Georgeanna See gar Jones, M.D. t Mason C. Andrews, M.D.t Anibal Acosta, M.D.t Clark Bundren, M.D. t Jairo Garcia, M.D.t Bruce Sandow, Ph.D.§ Lucinda Veeckt Charles Wilkes, M.D. t Jeannine Witmyerll J. Edward Wortham, Ph.D.11 George Wright, Ph.D.~ Eastern Virginia Medical School and Old Dominion University, Norfolk, Virginia
Several aspects of the program of in vitro fertilization (IVF), or, as it is called in Norfolk, the program for the Vital Initiation of Pregnancy (VIP), have been or are in the process of publication. However, because there has been no overall account, it seems appropriate to give a brief report of a general nature covering the period from the beginning of the effort in late February 1980 through December 31,1981. Although minor changes were constantly made in the protocol, there were two major revisions. Therefore, a discussion of the program during three distinct periods, i.e., 1980, 1981-Phase I, and 1981-Phase II, is necessary. During 1980 and 1981 all patients had either no fallopian tubes or irreparable tubes. Fertil Steril 38:14, 1982 1980
One should recall that at the beginning of 1980 there were no published details of a program for in vitro fertilization (IVF) that had resulted in the birth of a child. Received February 22,1982; accepted May 11, 1982. *Presented at the Thirty-Eighth Annual Meeting of The American Fertility Society, March 20 to 24,1982, Las Vegas, Nevada. tDepartment of Obstetrics and Gynecology, Eastern Virginia Medical School. tReprint requests: Howard W. Jones, Jr., M.D., Department of Obstetrics and Gynecology, Eastern Virginia Medical School, 603 Medical Tower, Norfolk, Virginia 23507. §Department of Anatomy, Eastern Virginia Medical School. IIDepartment of Biological Sciences, Old Dominion University. ~Department of Microbiology and Immunology .and Immunology Program, Eastern Virginia Medical School. 14
Jones et aI. In vitro fertilization at Norfolk
In February of 1979 Steptoe et al. presented their data before the Royal College of Obstetricians and Gynecologists in London. However, publication did not occur until late in 1980. 1 The report of the first pregnancy in Australia also appeared only in 1980. 2 Nevertheless, there were several reports of basic studies from both England and Australia and elsewhere. 3 -s From available information there was every reason for one to believe that clinical trials were timely and would pinpoint and improve the efficacy ofthe process. The Norfolk program greatly benefited from visits to and from the two active Australian programs in Melbourne, with the result that it was possible for us to utilize their very great experience. Based on available information, it seemed essential to provide for the initiation of a program Fertility and Sterility
that would (1) use the natural cycle instead of a stimulated controlled cycle, with which Steptoe et al. had failed\ (2) aspirate the single dominant follicle at the last possible moment prior to expected ovulation, since it was thought impossible to further "mature" an oocyte in vitro (such a concept required around-the-clock laparoscopic capability); (3) utilize a reliable method to predict the hour of ovulation; (4) utilize a proven aspiration technique; (5) take extreme measures to maintain a special environment for the oocyte (this included suitable temperature, low oxygen tension, suitable osmolality, suitable pH, etc.); and (6) provide a means of inseminating the egg with the least possible delay, within a few seconds if possible. We would refer to the program as Vital Initiation of Pregnancy, or VIP. We then decided to utilize a serum luteinizing hormone (LH) surge to predict ovulation. It was found that ovulation occurred after 28 hours after the beginning of the LH surge. 9 The aspiration requirement seemed to be fulfilled by the use of a needle with an inner diameter of 1.2 mm. 5 Carbon dioxide was used for the pneumoperitoneum for fear of an embolus from the 90% nitrous mixture recommended by Steptoe et al. 1 The results during 1980 were disappointing. From 41 laparoscopies only 19 (46%) fertilizable (preovulatory) eggs were obtained. During the year the yield was greatly improved by the use of a newly designed aspiration technique.lO Monitoring by television of each aspiration was also initiated. Of the 19 preovulatory eggs, 4 were utilized for an in vivo fertilization attempt whereby there was immediate transfer to the uterus without prior incubation or fertilization, with the hope that the sperm already provided in the uterus would fertilize the oocytes. Of the remaining 15 preovulatory eggs, fertilization was obtained in 9, and these were transferred to the uterus. There were no pregnancies in either the in vivo or in vitro series. These disappointing results for the normal cycle were shared by others. For example, at the Women's Hospital in Melbourne in 1980, 107 laparoscopies during the natural cycle were done with inefficiencies at each step of the procedure not too different from those at Norfolk and without a pregnancy.11 1981-PHASE I
Prior to the resumption of patient processing in 1981, the following protocol was adopted. Vol. 38, No.1, July 1982
1. In spite of the earlier British experience and advice, the natural cycle was abandoned in favor of stimulated controlled ovulation. The purpose of this was to obtain more than one fertilizable oocyte and to assure follicular maturation in a given cycle. The experience during 1980 with the natural cycle indicated that the stress of the situation often caused menstrual and follicular abnormalities. As a not undesirable adjunct, the use of controlled ovulation enabled definitive prior scheduling of the laparoscopic follicular aspirations. 2. For the stimulated controlled ovulations, human menopausal gonadotropin (hMG) (Pergonal, Serono Laboratories, Inc., Braintree, MA) was used in preference to clomiphene because it was anticipated that in a normal cycle the ovarian response would be more dependable with hMG than with clomiphene. Thus, with clomiphene in the experience of the Royal Woman's Hospital in Melbourne, 22% of cycles had to be passed without laparoscopy because of poor ovarian response. 11 Furthermore, prior personal experience-unpublished in regard to hMG but published in regard to clomiphene 12-suggested that the miscarriage rate was likely to be less with hMG than with Clomid (Merrell-National Laboratories, Cincinnati, OH). It is realized that others have had troublesome miscarriage rates after the use ofhMGP 3. Exogenous human chorionic gonadotropin (hCG) was used to substitute for the midcycle LH surge. It was observed that in the natural cycle hMG suppressed endogenous LH and a spontaneous LH surge did not occur at the expected time. 14, 15 Laparoscopy for follicular aspiration was scheduled 36 to 38 hours after hCG administration. The details for the use of hMG and administration ofhCG during 1981-Phase I is contained in a separate report. 16 4. The use of a realtime sector ultrasonograph to monitor follicular growth became routine. This was carried out each day and proved useful but never came to be considered the definitive measure of follicular development. This latter judgment was based partially on the difficulty of ultrasounding patients who were candidates for this program because they often had troublesome pelvic adhesions, retention cysts, stumps of tubes, and so on, which were difficult to interpret by ultrasonography. 5. Quality control in the laboratory was tightened. Jones et al. In vitro fertilization at Norfolk
15
whereas in 1981-Phase I cleavage was obtained in eggs from 12 of 26 cycles, or 46%. However, in these 12 cycles there were 16 eggs that cleaved, 1 in 9 cycles, 2 each in 2 cycles, and 3 in 1 cycle. During 1981-Phase I, two pregnancies occurred, each in a cycle during which only one cleaving egg was transferred. 1981-PHASE I PREGNANCIES VIP PREGNANCY 1
J.C., 4-14- 81, 8:45a.m. (Rt -Long.) Figure 1 Ultrasonographic study. 24 hours before aspiration. There seemed to be two well-developed but oval-shaped follicles in the right ovary. Eggs were obtained from each of these follicles.
6. Early in 1981 supplementary maturation in ~itro of the oocytes prior to insemination was initiated, at the suggestion of the group at the Royal Victoria Hospital in MelbourneP Although contrary to conventional wisdom, the concept was not unreasonable. After all,aspiration of . the follicle takes place prior to spontaneous ovulation and therefore presumably prior to complete maturation. The culture of the oocyte for 5 to 8 hours after harvesting presumably completes maturation and in practice seemed to increase the incidence of fertilization and development. During 1981-Phase I, there was improvement in aspiration of fertilizable oocytes in a given menstrual cycle, not only due to improved aspiration efficiency per follicle aspirated but also due to the availability of multiple follicles per cycle. Thus, in 1981-Phase I, fertilizable eggs were obtained in 26 of31 cycles (84%); whereas in 1980, with the spontaneous cycle, fertilizable eggs were obtained in only 19 of 41 (46%) of the cycles. In 1981-Phase I a total of 48 fertilizable eggs were obtained during the 26 cycles in which eggs were obtained. This is an average of 1.85 preovulatory eggs per cycle. The cleavage rate per cycle was not substantially different in 1981-Phase I from 1980. Thus, in 1980 the transfer rate was 60% of the single eggs obtained in 15 spontaneous cycles; 16
Jones et aI. In vitro fertilization at Norfolk
The patient was first seen January 21 , 1981', she had a birthdate of April 17 , 1953. She had had acute appendicitis with an emergency appendectomy at age 18. Six months after discontinuing oral contraception, she had a ruptured right ectopic pregnancy in September 1977. Four units of blood were required, and the right tube was removed with a cornual excision. In August 1978 there was a left ectopic pregnancy, which was removed from the left tube by a linear salpingostomy with repair. One unit of blood was required. In September 1980 there was a left ruptured tubal pregnancy with a left salpingectomy with a cornual excision. Four units of blood were required. A hysterosalpingogram (HSG) in December 1978 showed a normal uterine cavity. In January 1981 a screening laparoscopy showed that the right ovary was 100% available and the left ovary 60% available. Semen analysis showed 66.3 million sperm/ml with 72% morphologically normal and 52% quickly progressive. A Ureaplasma urealyticum culture was positive. The couple was treated with Vibramycin (Pfizer Inc., New York, NY) in February 1981.
Figure 2 Photomicrograph of the conceptus at the time of transfer. It is difficult to be precisely certain of the number of blastomeres' but by focusing up and down, we could see at least three and possibly four. Fertility and Sterility
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The patient had a menstrual period on April 6, 1981. hMG stimulation was started on April 8 with two ampules. She also received two ampules on April 9 and 10 and one on April 11. She received 10,000 IU ofhCG.on April 13. On April 14 ultrasonography showed two well-developed oval follicles in the right ovary (Fig. 1). This was her fourth daily ultrasonographic study. On April 15 (day 10 of menstrual cycle) laparoscopic aspiration of the two right follicles was carried out, and two preovulatory oocytes were obtained. In the left ovary there was a small follicle, which was aspirated; an immature oocyte was thus obtained. Cleavage occurred in only one of the preovulatory oocytes (Fig. 2), and the conceptus was transferred to the uterus, with the patient in the knee-chest position, on April 17 at 1:00 P.M. The luteal phase was .characterized by a remarkably high value for progesterone· (P) but with a fall that indicated that the luteal phase was destined to be short except for the intervention of the pregnancy. A serum J3-hCG of 20 mIU was obtained on day 13 after fertilization (Fig. 3). The J3-hCG and serum P values throughout pregnancy were within normal limits (Fig. 4). Ultrasonography was carried out nine times during the course of the pregnancy. The biparietal diameter was barely within the lower limits of normal throughout, but it continued to grow at a steady rate (Fig. 5). Fetal weight based on abdominal circumference was somewhat troubling. On December 21 it was estimated to be at 4.95 pounds, which was unchanged from a determination made on December 11. Serum alphafetoprotein (AFP) values were determined on 11 occasions between August 4, Vol. 38, No.1, July. 1982
1981, and November 23, 1981. All values were below the mean but.within 3 standard deviations (SD) below the mean. Serum estradiol (E 2 ) values were 13.2 pg/ml on November 24, 20.3 pg/ml on December 11, and 17 pg/ml on December 24. All these values were within normal limits. Non-stress tests at weekly intervals, beginning at 36 weeks' gestation, were within normal limits. Because of small fetal size and cessation of weight gain as determined by ultrasound and because oCa secondary concern because of prior bilateral cornual resections, it was elected to deliver the patient by cesarean section 9 days prior to term. A normal female infant weighing 5 lbs 12 oz with Apgar scores of 9 and 10 at 1 and 5 minutes, respectively, was delivered on December 28, 1981. Birth weight was regained at 6 days. The Brazelton neonatal behavior assessment was normal. VIP PREGNANCY 2
The patient was first seen March 19, 1981; she had.a birthdateof July 19, 1951. At age 21 she had had a right salpingectomy, and an incidental appendectomy for an acute abdominal episode, which proved to be due to pelvic inflammatory disease (PID). Because of distal tubal obstruction in the left tube, a cuff salpingostomy was done at age 23. The patient was divorced in 1974 and.remarried in 1978. During an infertility investigation an HSG showed a normal uterine cavity but no visualization of the remaining left tube. She was referred to the VIP program December 5,1980.
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The track of the beta subunit of hCG and P from the date of the LMP through delivery.
Jones .et aI. In vitro fertilization at Norfolk
17
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The pregnancy progressed normally, and a normal male infant was born on March 27, 1982.
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Figure 5 A chart of the biparietal diameter as determined by ultrasound during the last weeks of pregnancy. Although the biparietal diameter continued to grow, it was always at the lower limit of normal for the date.
A screening laparoscopy was carried out March 30, 1981, which revealed that the right ovary was 95% available and the left ovary 100% available. The left tube was a hydrosalpinx with peritubal adhesions. An injection of methylene blue through the cervix failed to enter the left tube. It was concluded that the tube was obstructed in two places and unsuitable for a second tuboplasty. The patient was admitted to our VIP program. A semen analysis revealed a volume of 4.7 ml with a count of 85.5 million sperm/ml with 33% quickly progressive and 76% normally formed. A culture for Ureaplasma urealyticum was negative. The patient had a menstrual period on June 28, 1981. She received two ampules of hMG on July 1, 2, and 3, and one ampule on July 4 and 5. Laparoscopy was carried out on July 9 with aspiration of three follicles and the harvesting of three eggs. One egg progressed to the four~cell stage and was transferred July 11 at 11:30 A.M. with the patient. in the knee-chest position. A serum~-hCG of 36 mIU was determined on July 21, i.e., the 13th day after aspiration (Fig. 6). 18
1981-PHASE II
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Jones et aI. In vitro fertilization at Norfolk
The program did not process any patients during the summer of 1981. The interval September 15 to December 15 was designated 1981-Phase II. On resuming operations in the fall, we initiated several changes in procedure. The 1981Phase II protocol comprised the following points: 1. Stimulated controlled ovulation was continued with the use of hMG and hCG. 2. Serum E2 values quickly determined became available on a daily basis, so that at 4:00 P.M. the hMG injections could be varied according to the serum E2 values at 8:00 A.M. The details of the indications for the initiation and discontinuation ofhMG as well as the indications for the injection of hCG are shown in Table 1. 3. There was further improvement in laboratory quality control. For example, extreme measures were taken to insure a water supply free of toxic substances. Water was glass-distilled at least four times. This water was used not only for various media but as a final rinse for laparoscopic equipment which might have contact with the oocyte. The pH of the insemination and development media was raised from 7.2 to 7.4. In addition, all media were pretested with a mouse oocyte system prior to use in the patient. Although there is evidence that has been interL.S. 100
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Figure 6 The track of E 2 , the beta subunit of heG, and P from the date of the LMP through day 30 of that cycle in VIP pregnancy 2.
Fertility and Sterility
subsequently have them accept fertilization and develop cleavage. This made it necessary to introduce a new classification of oocytes as obtained from the follicle. In addition to preovulatory, immature, and atretic oocytes, a group referred to as immature matured in vitro (lMIV) was added, so that if an immature oocyte became fertilized, it was classified as IMIV; thus, fertilizable oocytes include preovulatory oocytes and IMIVs. With these changes, results during 1981Phase II were substantially better than earlier. Thus, in 24 laparoscopic cycles fertilizable eggs were obtained in 22, or 92%. The only cycle during which an egg was not obtained was one where there was technical difficulty with the aspiration needle, which had an unrecognized burr on the tip that caused follicular tearing. In one other cycle only immature eggs were obtained. Eggs were fertilized -from 21 of the 22 cycles during which fertilizable eggs were obtained, and transfers occurred in 19 cycles. There were five pregnancies, the outcomes of which are not determinable at the time of writing (Table 2). Utilizing the data from the 311981 cycles with transfers, the importance of transferring more than one conceptus becomes evident. Thus, with the transfer of a single conceptus the pregnancy rate was 13%, with two, 31%, and with three, 50%. These numbers are small and require confirmation, but the trend is impressive (Table 3).
Table 1. hMG-hCG Protocol General 1. The clinical parameters, blood for E2 and any other assays, and ultrasound are performed at approximately 8:00A.M. 2. All medications such as hMG should be given at about 4:00 P.M. so that advantage can be taken of the assays performed on the 8:00 A.M. blood. 3. hCG should be given at 6:00 P.M. with a laparoscopy posted at 7:30 A.M., 37% hours later, so that by the time the egg is harvested it will be more or less 38 hours old. Day 3 (day 5 if a long cycle) 8:00 A.M. Clinical parameters, blood, and ultrasound. 4:00 P.M. Two ampules ofhMG. Days 4 and 5 8:00 A.M. Clinical parameters and ultrasound mayor may not be repeated, depending on the clinical situation, but no blood need be taken on these days. 4:00 P.M. Two ampules of hMG. Day 6 and each day until hMG is discontinued 8:00 A.M. Clinical parameters, blood, and ultrasound. 4:00 P.M. Two ampules of hMG. Indications for the discontinuation of hMG hMG should be discontinued on the day when the E2 is 300 pg/ml or more and the clinical parameters have shifted for . at least 1 day or E2 reaches 600 pg/ml without a shift in clinical parameters or the clinical parameters have shifted for 3 consecutive days regardless of the level of the E2 or by ultrasound study the enlargement of the largest follicle has been steady and has reached 14 mm in diameter. Indications for administration of hCG The hCG will be given 50 hours after the last hMG. General Clinical parameters, blood, and ultrasound to be continued at 8:00 A.M. daily through the day prior to laparoscopy, beginning with day 6. On the day oflaparoscopy, blood will be drawn at the time oflaparoscopy for such assays as seem appropriate.
DISCUSSION
The variables in a program of IVF are legion, and the fertilizable oocytes are limited and precious. Therefore, it is not feasible to design an experiment to test each variable. These circumstances have made it necessary for those in such programs to develop a procedure based on evidence that is less than conclusive. In the program at Norfolk it has developed that many of the original assumptions were incorrect. Thus, the stimulated cycle has turned out to be useful for the harvesting of oocytes. Whether
preted to indicate that light might not be beneficiaI,18,19 photomicrographs were routinely taken. 4. The concept of supplementary oocyte maturation in vitro was continued and extended. We were able to incubate prior to insemination 00cytes that seemed morphologically immature and Table 2. Results
Phase 1-1981
1980
A B C D E
Laparoscopies Fertilizable eggs Eggs fertilized Transfers Pregnancies aND, not determined.
Vol. 38, No.1, July 1982
No.
%A
41 19 NDa 13 0
46 ND 32 0
%8
ND 68 0
%C
ND ND
%D
No.
%A
0
31 26 ND 12 2
84 ND 39 6
%8
ND 46 8
%C
ND ND
Phase 11-1981
%D
No.
%A
%8
%C
%D
17
24 22 21 19 5
92 88 79 21
95 86 23
90 24
26
Jones et al. In vitro fertilization at Norfolk
19
Table 3. 1981-31 Transfers Single conceptus Pregnancies Double concepti Pregnancies Triple concepti Pregnancies Quadruple concepti Pregnancies
15 2 (13%) 13 4 (31%) 2 1 (50%) 1
o
hMG is better for this purpose than clomiphene remains to be determined. Nevertheless, the use of some stimulating agent in the obtaining of more than one fertilizable oocyte per cycle offers the possibility of exceeding the normal expectation of pregnancy per cycle. The availability of more than one oocyte per cycle greatly improves the efficiency of each of the steps in the program. Among other incorrect assumptions was the concept that supplementary incubation in vitro could not materially further mature an immature oocyte. Accumulated experience at the Norfolk program and elsewhere 17 indicates that supplementary incubation prior to fertilization is very beneficial, even when the aspirated oocyte seems to be preovulatory by morphologic criteria. In at least one instance (data not reported earlier) an oocyte with a germinal vesicle was observed to break down the germinal vesicle and extrude the first polar body after 26 hours in incubation. This same oocyte was then fertilized and developed into a five-cell conceptus within 44 hours. Thus, supplementary oocyte incubation prior to incubation seems to be a useful strategy. The length of the incubation for supplemental maturation has been 5 to 8 hours for preovulatory oocytes, but the upper limit of this supplementary maturation for less mature oocytes has yet to be determined. Because ofthe possibility of in vitro maturation, the preovulatory window of time through which an oocyte must be obtained seems wider than previously thought. There were other misconceptions. Sufficient light to photograph the oocyte and early conceptus certainly does not necessarily preclude cleavage. Pending evidence which might indicate the contrary, there is no plan to alter routine photography. Despite a lack of evidence, there has been an expression of concern that ultrasonography might be detrimental. In the above series all pregnant patients were subject to a minimum of four examinations by ultrasound prior to ovulation, and pregnant uteri were examined up to nine times during pregnancy with no detectable or recognizable damage. 20
Jones et aI. In vitro fertilization at Norfolk
The highest type of internal quality control in the laboratory is an essential prerequisite. It would seem that trace amounts of toxic substances must be eliminated. It may be extreme to require that all media support cleavage to the blastocyst stage in early mouse concepti, but an occasional batch of freshly made medium has been discarded because of failure to do so. Finally, while the main object of a program of IVF is to overcome a previously untreatable barrier to fertility, the opportunity provided by such a program to investigate many aspects of early reproduction, such as the ultimate details of follicular maturation, sperm-oocyte interaction, and early cleavage, are breathtaking and await development.
REFERENCES 1. Steptoe PC, Edwards RG, Purdy JM: Clinical aspects of pregnancies established with cleaving embryos grown in vitro. Br J Obstet Gynaecol 87:757, 1980 2. Lopata A, Johnston IWH, Hoult IJ, Speirs AI: Pregnancy following intrauterine implantation of an embryo obtained by in vitro fertilization of a preovulatory egg. Fertil Steril 33:117, 1980 3. Edwards RG: Maturation in vitro of human ovarian 00cytes. Lancet 2:926, 1965 4. Edwards RG, Donahue RP, Baramki TA, Jones HW Jr: Preliminary attempts to fertilize human oocytes matured in vitro. Am J Obstet Gynecol 96:192, 1966 5. Steptoe PC, Edwards RG: Laparoscopic recovery of preovulatory human oocytes after priming of ovaries with gonadotropins. Lancet 1:683, 1970 6. Steptoe PC, Edwards RG, Purdy JM: Human blastocysts grown in culture. Nature 229:132, 1971 7. Edwards RG: Studies on human conception. Am J Obstet GynecoI117:587,1973 8. Soupart P: In vitro fertilization and embryo transfer. In Current Problems in 'Obstetrics and Gynecology, Vol 3. Chicago, Yearbook Medical Publishers, October-November 1979 . 9. Garcia JE, Jones GS, Wright GL Jr: Prediction of the time of ovulation. Fertil Steril 36:308, 1981 10. Jones HW Jr, Acosta AA, Garcia J: A technique for the aspiration of oocytes from human ovarian follicles. Fertil Steril 37:26, 1982 11. Johnston I, Lopata A, Speirs A, Hoult I, Kellow G, duPlessis Y: In vitro fertilization: the challenge of the eighties. Fertil Steril 36:699, 1981 12. Garcia J, Jones GS: The use of clomiphene citrate. Fertil Steril 28:707, 1977 13. Ben-Rafael Z, Mashiach S, Oelsner G, Farine D, Lunenfeld B, Serr DM: Spontaneous pregnancy and its outcome after human menopausal gonadotropinlhuman chorionic gonadotropin-induced pregnancy. Fertil Steril 36:560, 1981 14. Lenton EA, Adams M, Cooke ID: Plasma steroid and gonadotropin profiles in ovulatory but infertile women. Clin Endocrinol 8:241, 1978
Fertility and Sterility
15. Schoemaker J, Wentz A, Jones GS: Induction of ovulation with pure "FSH" in patients with amenorrhea and elevated LH levels. Fertil Steril 28:295, 1977 16. Garcia J, Jones GS: Relation of the mid-cycle FSH surge to fertility. In press 17. Trounson AO, Mohr LH, Wood C, Leeton JF: In vitro fertilization, culture and transfer of human embryos: effect of delayed insemination. In press
Vol. 38, No.1, July 1982
18. Daniel JC: Cleavage of mammalian ova inhibited by visible light. Nature 201:317,1964 19. Hirao Y, Yanagimachi R: Detrimental effects of visible light on meiosis of mammalian eggs in vitro. J Exp Zool 206:365, 1978
Jones et al. In vitro fertilization at Norfolk
21
FERTILITY AND STERILITY Copyright 0 1982 The American Fertility Society
Printed in U.SA.
The program for in vitro fertilization at Norfolk*
Howard W. Jones, Jr., M.D.t:J: Georgeanna See gar Jones, M.D. t Mason C. Andrews, M.D.t Anibal Acosta, M.D.t Clark Bundren, M.D. t Jairo Garcia, M.D.t Bruce Sandow, Ph.D.§ Lucinda Veeckt Charles Wilkes, M.D. t Jeannine Witmyerll J. Edward Wortham, Ph.D.11 George Wright, Ph.D.~ Eastern Virginia Medical School and Old Dominion University, Norfolk, Virginia
Several aspects of the program of in vitro fertilization (IVF), or, as it is called in Norfolk, the program for the Vital Initiation of Pregnancy (VIP), have been or are in the process of publication. However, because there has been no overall account, it seems appropriate to give a brief report of a general nature covering the period from the beginning of the effort in late February 1980 through December 31,1981. Although minor changes were constantly made in the protocol, there were two major revisions. Therefore, a discussion of the program during three distinct periods, i.e., 1980, 1981-Phase I, and 1981-Phase II, is necessary. During 1980 and 1981 all patients had either no fallopian tubes or irreparable tubes. Fertil Steril 38:14, 1982 1980
One should recall that at the beginning of 1980 there were no published details of a program for in vitro fertilization (IVF) that had resulted in the birth of a child. Received February 22,1982; accepted May 11, 1982. *Presented at the Thirty-Eighth Annual Meeting of The American Fertility Society, March 20 to 24,1982, Las Vegas, Nevada. tDepartment of Obstetrics and Gynecology, Eastern Virginia Medical School. tReprint requests: Howard W. Jones, Jr., M.D., Department of Obstetrics and Gynecology, Eastern Virginia Medical School, 603 Medical Tower, Norfolk, Virginia 23507. §Department of Anatomy, Eastern Virginia Medical School. IIDepartment of Biological Sciences, Old Dominion University. ~Department of Microbiology and Immunology .and Immunology Program, Eastern Virginia Medical School. 14
Jones et aI. In vitro fertilization at Norfolk
In February of 1979 Steptoe et al. presented their data before the Royal College of Obstetricians and Gynecologists in London. However, publication did not occur until late in 1980. 1 The report of the first pregnancy in Australia also appeared only in 1980. 2 Nevertheless, there were several reports of basic studies from both England and Australia and elsewhere. 3 -s From available information there was every reason for one to believe that clinical trials were timely and would pinpoint and improve the efficacy ofthe process. The Norfolk program greatly benefited from visits to and from the two active Australian programs in Melbourne, with the result that it was possible for us to utilize their very great experience. Based on available information, it seemed essential to provide for the initiation of a program Fertility and Sterility
that would (1) use the natural cycle instead of a stimulated controlled cycle, with which Steptoe et al. had failed\ (2) aspirate the single dominant follicle at the last possible moment prior to expected ovulation, since it was thought impossible to further "mature" an oocyte in vitro (such a concept required around-the-clock laparoscopic capability); (3) utilize a reliable method to predict the hour of ovulation; (4) utilize a proven aspiration technique; (5) take extreme measures to maintain a special environment for the oocyte (this included suitable temperature, low oxygen tension, suitable osmolality, suitable pH, etc.); and (6) provide a means of inseminating the egg with the least possible delay, within a few seconds if possible. We would refer to the program as Vital Initiation of Pregnancy, or VIP. We then decided to utilize a serum luteinizing hormone (LH) surge to predict ovulation. It was found that ovulation occurred after 28 hours after the beginning of the LH surge. 9 The aspiration requirement seemed to be fulfilled by the use of a needle with an inner diameter of 1.2 mm. 5 Carbon dioxide was used for the pneumoperitoneum for fear of an embolus from the 90% nitrous mixture recommended by Steptoe et al. 1 The results during 1980 were disappointing. From 41 laparoscopies only 19 (46%) fertilizable (preovulatory) eggs were obtained. During the year the yield was greatly improved by the use of a newly designed aspiration technique.lO Monitoring by television of each aspiration was also initiated. Of the 19 preovulatory eggs, 4 were utilized for an in vivo fertilization attempt whereby there was immediate transfer to the uterus without prior incubation or fertilization, with the hope that the sperm already provided in the uterus would fertilize the oocytes. Of the remaining 15 preovulatory eggs, fertilization was obtained in 9, and these were transferred to the uterus. There were no pregnancies in either the in vivo or in vitro series. These disappointing results for the normal cycle were shared by others. For example, at the Women's Hospital in Melbourne in 1980, 107 laparoscopies during the natural cycle were done with inefficiencies at each step of the procedure not too different from those at Norfolk and without a pregnancy.11 1981-PHASE I
Prior to the resumption of patient processing in 1981, the following protocol was adopted. Vol. 38, No.1, July 1982
1. In spite of the earlier British experience and advice, the natural cycle was abandoned in favor of stimulated controlled ovulation. The purpose of this was to obtain more than one fertilizable oocyte and to assure follicular maturation in a given cycle. The experience during 1980 with the natural cycle indicated that the stress of the situation often caused menstrual and follicular abnormalities. As a not undesirable adjunct, the use of controlled ovulation enabled definitive prior scheduling of the laparoscopic follicular aspirations. 2. For the stimulated controlled ovulations, human menopausal gonadotropin (hMG) (Pergonal, Serono Laboratories, Inc., Braintree, MA) was used in preference to clomiphene because it was anticipated that in a normal cycle the ovarian response would be more dependable with hMG than with clomiphene. Thus, with clomiphene in the experience of the Royal Woman's Hospital in Melbourne, 22% of cycles had to be passed without laparoscopy because of poor ovarian response. 11 Furthermore, prior personal experience-unpublished in regard to hMG but published in regard to clomiphene 12-suggested that the miscarriage rate was likely to be less with hMG than with Clomid (Merrell-National Laboratories, Cincinnati, OH). It is realized that others have had troublesome miscarriage rates after the use ofhMGP 3. Exogenous human chorionic gonadotropin (hCG) was used to substitute for the midcycle LH surge. It was observed that in the natural cycle hMG suppressed endogenous LH and a spontaneous LH surge did not occur at the expected time. 14, 15 Laparoscopy for follicular aspiration was scheduled 36 to 38 hours after hCG administration. The details for the use of hMG and administration ofhCG during 1981-Phase I is contained in a separate report. 16 4. The use of a realtime sector ultrasonograph to monitor follicular growth became routine. This was carried out each day and proved useful but never came to be considered the definitive measure of follicular development. This latter judgment was based partially on the difficulty of ultrasounding patients who were candidates for this program because they often had troublesome pelvic adhesions, retention cysts, stumps of tubes, and so on, which were difficult to interpret by ultrasonography. 5. Quality control in the laboratory was tightened. Jones et al. In vitro fertilization at Norfolk
15
whereas in 1981-Phase I cleavage was obtained in eggs from 12 of 26 cycles, or 46%. However, in these 12 cycles there were 16 eggs that cleaved, 1 in 9 cycles, 2 each in 2 cycles, and 3 in 1 cycle. During 1981-Phase I, two pregnancies occurred, each in a cycle during which only one cleaving egg was transferred. 1981-PHASE I PREGNANCIES VIP PREGNANCY 1
J.C., 4-14- 81, 8:45a.m. (Rt -Long.) Figure 1 Ultrasonographic study. 24 hours before aspiration. There seemed to be two well-developed but oval-shaped follicles in the right ovary. Eggs were obtained from each of these follicles.
6. Early in 1981 supplementary maturation in ~itro of the oocytes prior to insemination was initiated, at the suggestion of the group at the Royal Victoria Hospital in MelbourneP Although contrary to conventional wisdom, the concept was not unreasonable. After all,aspiration of . the follicle takes place prior to spontaneous ovulation and therefore presumably prior to complete maturation. The culture of the oocyte for 5 to 8 hours after harvesting presumably completes maturation and in practice seemed to increase the incidence of fertilization and development. During 1981-Phase I, there was improvement in aspiration of fertilizable oocytes in a given menstrual cycle, not only due to improved aspiration efficiency per follicle aspirated but also due to the availability of multiple follicles per cycle. Thus, in 1981-Phase I, fertilizable eggs were obtained in 26 of31 cycles (84%); whereas in 1980, with the spontaneous cycle, fertilizable eggs were obtained in only 19 of 41 (46%) of the cycles. In 1981-Phase I a total of 48 fertilizable eggs were obtained during the 26 cycles in which eggs were obtained. This is an average of 1.85 preovulatory eggs per cycle. The cleavage rate per cycle was not substantially different in 1981-Phase I from 1980. Thus, in 1980 the transfer rate was 60% of the single eggs obtained in 15 spontaneous cycles; 16
Jones et aI. In vitro fertilization at Norfolk
The patient was first seen January 21 , 1981', she had a birthdate of April 17 , 1953. She had had acute appendicitis with an emergency appendectomy at age 18. Six months after discontinuing oral contraception, she had a ruptured right ectopic pregnancy in September 1977. Four units of blood were required, and the right tube was removed with a cornual excision. In August 1978 there was a left ectopic pregnancy, which was removed from the left tube by a linear salpingostomy with repair. One unit of blood was required. In September 1980 there was a left ruptured tubal pregnancy with a left salpingectomy with a cornual excision. Four units of blood were required. A hysterosalpingogram (HSG) in December 1978 showed a normal uterine cavity. In January 1981 a screening laparoscopy showed that the right ovary was 100% available and the left ovary 60% available. Semen analysis showed 66.3 million sperm/ml with 72% morphologically normal and 52% quickly progressive. A Ureaplasma urealyticum culture was positive. The couple was treated with Vibramycin (Pfizer Inc., New York, NY) in February 1981.
Figure 2 Photomicrograph of the conceptus at the time of transfer. It is difficult to be precisely certain of the number of blastomeres' but by focusing up and down, we could see at least three and possibly four. Fertility and Sterility
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Figure 3 The track of E 2 , the beta subunit of hCG, and P from the last menstrual period (LMP) through day 28 of that cycle.
The patient had a menstrual period on April 6, 1981. hMG stimulation was started on April 8 with two ampules. She also received two ampules on April 9 and 10 and one on April 11. She received 10,000 IU ofhCG.on April 13. On April 14 ultrasonography showed two well-developed oval follicles in the right ovary (Fig. 1). This was her fourth daily ultrasonographic study. On April 15 (day 10 of menstrual cycle) laparoscopic aspiration of the two right follicles was carried out, and two preovulatory oocytes were obtained. In the left ovary there was a small follicle, which was aspirated; an immature oocyte was thus obtained. Cleavage occurred in only one of the preovulatory oocytes (Fig. 2), and the conceptus was transferred to the uterus, with the patient in the knee-chest position, on April 17 at 1:00 P.M. The luteal phase was .characterized by a remarkably high value for progesterone· (P) but with a fall that indicated that the luteal phase was destined to be short except for the intervention of the pregnancy. A serum J3-hCG of 20 mIU was obtained on day 13 after fertilization (Fig. 3). The J3-hCG and serum P values throughout pregnancy were within normal limits (Fig. 4). Ultrasonography was carried out nine times during the course of the pregnancy. The biparietal diameter was barely within the lower limits of normal throughout, but it continued to grow at a steady rate (Fig. 5). Fetal weight based on abdominal circumference was somewhat troubling. On December 21 it was estimated to be at 4.95 pounds, which was unchanged from a determination made on December 11. Serum alphafetoprotein (AFP) values were determined on 11 occasions between August 4, Vol. 38, No.1, July. 1982
1981, and November 23, 1981. All values were below the mean but.within 3 standard deviations (SD) below the mean. Serum estradiol (E 2 ) values were 13.2 pg/ml on November 24, 20.3 pg/ml on December 11, and 17 pg/ml on December 24. All these values were within normal limits. Non-stress tests at weekly intervals, beginning at 36 weeks' gestation, were within normal limits. Because of small fetal size and cessation of weight gain as determined by ultrasound and because oCa secondary concern because of prior bilateral cornual resections, it was elected to deliver the patient by cesarean section 9 days prior to term. A normal female infant weighing 5 lbs 12 oz with Apgar scores of 9 and 10 at 1 and 5 minutes, respectively, was delivered on December 28, 1981. Birth weight was regained at 6 days. The Brazelton neonatal behavior assessment was normal. VIP PREGNANCY 2
The patient was first seen March 19, 1981; she had.a birthdateof July 19, 1951. At age 21 she had had a right salpingectomy, and an incidental appendectomy for an acute abdominal episode, which proved to be due to pelvic inflammatory disease (PID). Because of distal tubal obstruction in the left tube, a cuff salpingostomy was done at age 23. The patient was divorced in 1974 and.remarried in 1978. During an infertility investigation an HSG showed a normal uterine cavity but no visualization of the remaining left tube. She was referred to the VIP program December 5,1980.
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The track of the beta subunit of hCG and P from the date of the LMP through delivery.
Jones .et aI. In vitro fertilization at Norfolk
17
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The pregnancy progressed normally, and a normal male infant was born on March 27, 1982.
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Figure 5 A chart of the biparietal diameter as determined by ultrasound during the last weeks of pregnancy. Although the biparietal diameter continued to grow, it was always at the lower limit of normal for the date.
A screening laparoscopy was carried out March 30, 1981, which revealed that the right ovary was 95% available and the left ovary 100% available. The left tube was a hydrosalpinx with peritubal adhesions. An injection of methylene blue through the cervix failed to enter the left tube. It was concluded that the tube was obstructed in two places and unsuitable for a second tuboplasty. The patient was admitted to our VIP program. A semen analysis revealed a volume of 4.7 ml with a count of 85.5 million sperm/ml with 33% quickly progressive and 76% normally formed. A culture for Ureaplasma urealyticum was negative. The patient had a menstrual period on June 28, 1981. She received two ampules of hMG on July 1, 2, and 3, and one ampule on July 4 and 5. Laparoscopy was carried out on July 9 with aspiration of three follicles and the harvesting of three eggs. One egg progressed to the four~cell stage and was transferred July 11 at 11:30 A.M. with the patient. in the knee-chest position. A serum~-hCG of 36 mIU was determined on July 21, i.e., the 13th day after aspiration (Fig. 6). 18
1981-PHASE II
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Jones et aI. In vitro fertilization at Norfolk
The program did not process any patients during the summer of 1981. The interval September 15 to December 15 was designated 1981-Phase II. On resuming operations in the fall, we initiated several changes in procedure. The 1981Phase II protocol comprised the following points: 1. Stimulated controlled ovulation was continued with the use of hMG and hCG. 2. Serum E2 values quickly determined became available on a daily basis, so that at 4:00 P.M. the hMG injections could be varied according to the serum E2 values at 8:00 A.M. The details of the indications for the initiation and discontinuation ofhMG as well as the indications for the injection of hCG are shown in Table 1. 3. There was further improvement in laboratory quality control. For example, extreme measures were taken to insure a water supply free of toxic substances. Water was glass-distilled at least four times. This water was used not only for various media but as a final rinse for laparoscopic equipment which might have contact with the oocyte. The pH of the insemination and development media was raised from 7.2 to 7.4. In addition, all media were pretested with a mouse oocyte system prior to use in the patient. Although there is evidence that has been interL.S. 100
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Figure 6 The track of E 2 , the beta subunit of heG, and P from the date of the LMP through day 30 of that cycle in VIP pregnancy 2.
Fertility and Sterility
subsequently have them accept fertilization and develop cleavage. This made it necessary to introduce a new classification of oocytes as obtained from the follicle. In addition to preovulatory, immature, and atretic oocytes, a group referred to as immature matured in vitro (lMIV) was added, so that if an immature oocyte became fertilized, it was classified as IMIV; thus, fertilizable oocytes include preovulatory oocytes and IMIVs. With these changes, results during 1981Phase II were substantially better than earlier. Thus, in 24 laparoscopic cycles fertilizable eggs were obtained in 22, or 92%. The only cycle during which an egg was not obtained was one where there was technical difficulty with the aspiration needle, which had an unrecognized burr on the tip that caused follicular tearing. In one other cycle only immature eggs were obtained. Eggs were fertilized -from 21 of the 22 cycles during which fertilizable eggs were obtained, and transfers occurred in 19 cycles. There were five pregnancies, the outcomes of which are not determinable at the time of writing (Table 2). Utilizing the data from the 311981 cycles with transfers, the importance of transferring more than one conceptus becomes evident. Thus, with the transfer of a single conceptus the pregnancy rate was 13%, with two, 31%, and with three, 50%. These numbers are small and require confirmation, but the trend is impressive (Table 3).
Table 1. hMG-hCG Protocol General 1. The clinical parameters, blood for E2 and any other assays, and ultrasound are performed at approximately 8:00A.M. 2. All medications such as hMG should be given at about 4:00 P.M. so that advantage can be taken of the assays performed on the 8:00 A.M. blood. 3. hCG should be given at 6:00 P.M. with a laparoscopy posted at 7:30 A.M., 37% hours later, so that by the time the egg is harvested it will be more or less 38 hours old. Day 3 (day 5 if a long cycle) 8:00 A.M. Clinical parameters, blood, and ultrasound. 4:00 P.M. Two ampules ofhMG. Days 4 and 5 8:00 A.M. Clinical parameters and ultrasound mayor may not be repeated, depending on the clinical situation, but no blood need be taken on these days. 4:00 P.M. Two ampules of hMG. Day 6 and each day until hMG is discontinued 8:00 A.M. Clinical parameters, blood, and ultrasound. 4:00 P.M. Two ampules of hMG. Indications for the discontinuation of hMG hMG should be discontinued on the day when the E2 is 300 pg/ml or more and the clinical parameters have shifted for . at least 1 day or E2 reaches 600 pg/ml without a shift in clinical parameters or the clinical parameters have shifted for 3 consecutive days regardless of the level of the E2 or by ultrasound study the enlargement of the largest follicle has been steady and has reached 14 mm in diameter. Indications for administration of hCG The hCG will be given 50 hours after the last hMG. General Clinical parameters, blood, and ultrasound to be continued at 8:00 A.M. daily through the day prior to laparoscopy, beginning with day 6. On the day oflaparoscopy, blood will be drawn at the time oflaparoscopy for such assays as seem appropriate.
DISCUSSION
The variables in a program of IVF are legion, and the fertilizable oocytes are limited and precious. Therefore, it is not feasible to design an experiment to test each variable. These circumstances have made it necessary for those in such programs to develop a procedure based on evidence that is less than conclusive. In the program at Norfolk it has developed that many of the original assumptions were incorrect. Thus, the stimulated cycle has turned out to be useful for the harvesting of oocytes. Whether
preted to indicate that light might not be beneficiaI,18,19 photomicrographs were routinely taken. 4. The concept of supplementary oocyte maturation in vitro was continued and extended. We were able to incubate prior to insemination 00cytes that seemed morphologically immature and Table 2. Results
Phase 1-1981
1980
A B C D E
Laparoscopies Fertilizable eggs Eggs fertilized Transfers Pregnancies aND, not determined.
Vol. 38, No.1, July 1982
No.
%A
41 19 NDa 13 0
46 ND 32 0
%8
ND 68 0
%C
ND ND
%D
No.
%A
0
31 26 ND 12 2
84 ND 39 6
%8
ND 46 8
%C
ND ND
Phase 11-1981
%D
No.
%A
%8
%C
%D
17
24 22 21 19 5
92 88 79 21
95 86 23
90 24
26
Jones et al. In vitro fertilization at Norfolk
19
Table 3. 1981-31 Transfers Single conceptus Pregnancies Double concepti Pregnancies Triple concepti Pregnancies Quadruple concepti Pregnancies
15 2 (13%) 13 4 (31%) 2 1 (50%) 1
o
hMG is better for this purpose than clomiphene remains to be determined. Nevertheless, the use of some stimulating agent in the obtaining of more than one fertilizable oocyte per cycle offers the possibility of exceeding the normal expectation of pregnancy per cycle. The availability of more than one oocyte per cycle greatly improves the efficiency of each of the steps in the program. Among other incorrect assumptions was the concept that supplementary incubation in vitro could not materially further mature an immature oocyte. Accumulated experience at the Norfolk program and elsewhere 17 indicates that supplementary incubation prior to fertilization is very beneficial, even when the aspirated oocyte seems to be preovulatory by morphologic criteria. In at least one instance (data not reported earlier) an oocyte with a germinal vesicle was observed to break down the germinal vesicle and extrude the first polar body after 26 hours in incubation. This same oocyte was then fertilized and developed into a five-cell conceptus within 44 hours. Thus, supplementary oocyte incubation prior to incubation seems to be a useful strategy. The length of the incubation for supplemental maturation has been 5 to 8 hours for preovulatory oocytes, but the upper limit of this supplementary maturation for less mature oocytes has yet to be determined. Because ofthe possibility of in vitro maturation, the preovulatory window of time through which an oocyte must be obtained seems wider than previously thought. There were other misconceptions. Sufficient light to photograph the oocyte and early conceptus certainly does not necessarily preclude cleavage. Pending evidence which might indicate the contrary, there is no plan to alter routine photography. Despite a lack of evidence, there has been an expression of concern that ultrasonography might be detrimental. In the above series all pregnant patients were subject to a minimum of four examinations by ultrasound prior to ovulation, and pregnant uteri were examined up to nine times during pregnancy with no detectable or recognizable damage. 20
Jones et aI. In vitro fertilization at Norfolk
The highest type of internal quality control in the laboratory is an essential prerequisite. It would seem that trace amounts of toxic substances must be eliminated. It may be extreme to require that all media support cleavage to the blastocyst stage in early mouse concepti, but an occasional batch of freshly made medium has been discarded because of failure to do so. Finally, while the main object of a program of IVF is to overcome a previously untreatable barrier to fertility, the opportunity provided by such a program to investigate many aspects of early reproduction, such as the ultimate details of follicular maturation, sperm-oocyte interaction, and early cleavage, are breathtaking and await development.
REFERENCES 1. Steptoe PC, Edwards RG, Purdy JM: Clinical aspects of pregnancies established with cleaving embryos grown in vitro. Br J Obstet Gynaecol 87:757, 1980 2. Lopata A, Johnston IWH, Hoult IJ, Speirs AI: Pregnancy following intrauterine implantation of an embryo obtained by in vitro fertilization of a preovulatory egg. Fertil Steril 33:117, 1980 3. Edwards RG: Maturation in vitro of human ovarian 00cytes. Lancet 2:926, 1965 4. Edwards RG, Donahue RP, Baramki TA, Jones HW Jr: Preliminary attempts to fertilize human oocytes matured in vitro. Am J Obstet Gynecol 96:192, 1966 5. Steptoe PC, Edwards RG: Laparoscopic recovery of preovulatory human oocytes after priming of ovaries with gonadotropins. Lancet 1:683, 1970 6. Steptoe PC, Edwards RG, Purdy JM: Human blastocysts grown in culture. Nature 229:132, 1971 7. Edwards RG: Studies on human conception. Am J Obstet GynecoI117:587,1973 8. Soupart P: In vitro fertilization and embryo transfer. In Current Problems in 'Obstetrics and Gynecology, Vol 3. Chicago, Yearbook Medical Publishers, October-November 1979 . 9. Garcia JE, Jones GS, Wright GL Jr: Prediction of the time of ovulation. Fertil Steril 36:308, 1981 10. Jones HW Jr, Acosta AA, Garcia J: A technique for the aspiration of oocytes from human ovarian follicles. Fertil Steril 37:26, 1982 11. Johnston I, Lopata A, Speirs A, Hoult I, Kellow G, duPlessis Y: In vitro fertilization: the challenge of the eighties. Fertil Steril 36:699, 1981 12. Garcia J, Jones GS: The use of clomiphene citrate. Fertil Steril 28:707, 1977 13. Ben-Rafael Z, Mashiach S, Oelsner G, Farine D, Lunenfeld B, Serr DM: Spontaneous pregnancy and its outcome after human menopausal gonadotropinlhuman chorionic gonadotropin-induced pregnancy. Fertil Steril 36:560, 1981 14. Lenton EA, Adams M, Cooke ID: Plasma steroid and gonadotropin profiles in ovulatory but infertile women. Clin Endocrinol 8:241, 1978
Fertility and Sterility
15. Schoemaker J, Wentz A, Jones GS: Induction of ovulation with pure "FSH" in patients with amenorrhea and elevated LH levels. Fertil Steril 28:295, 1977 16. Garcia J, Jones GS: Relation of the mid-cycle FSH surge to fertility. In press 17. Trounson AO, Mohr LH, Wood C, Leeton JF: In vitro fertilization, culture and transfer of human embryos: effect of delayed insemination. In press
Vol. 38, No.1, July 1982
18. Daniel JC: Cleavage of mammalian ova inhibited by visible light. Nature 201:317,1964 19. Hirao Y, Yanagimachi R: Detrimental effects of visible light on meiosis of mammalian eggs in vitro. J Exp Zool 206:365, 1978
Jones et al. In vitro fertilization at Norfolk
21